Nucleic acids encoding ribonucleases and methods of making them

ABSTRACT

pET11d-rOnc(Q1, M23L) DNA is subjected to two different site-directed mutations, each using an overlapping PCR protocol. One of the site-directed mutations changes the amino acid residue at position 23 of the encoded protein from leucine to methionine, whereby the encoded protein can be made into ranpirnase by cleaving the N-terminal methionine residue and allowing the adjacent glutamine residue to autocyclize. The other site-directed mutation changes the amino acid residue at position 72 of the encoded protein from serine to cysteine, thereby producing an encoded protein that can be made into a cysteinized ranpirnase by cleaving the N-terminal methionine residue and allowing the adjacent glutamine residue to autocyclize.

BACKGROUND OF THE INVENTION

The invention relates to Ribonucleases (RNases), and more particularlyrelates to ranpirnase. In its most immediate sense, the inventionrelates to nucleic acids that encode proteins that can be used toproduce ranpirnase and an RNase that is highly homologous to it.

Ranpirnase is the generic name of an RNase that is produced by AlfacellCorporation (assignee herein) under the registered trademark ONCONASE.Ranpirnase is a protein 104 residues long, with a blocked N-terminal ofpyroglutamic acid (<Glu) that is produced by autocyclization ofglutamine (Gln). It is disclosed in U.S. Pat. No. 5,559,212. As isstated therein, ranpirnase is presently produced from eggs of the ranapipiens frog. It would be advantageous to produce ranpirnase usingrecombinant DNA technology instead of processing biological material.

Additionally, work done at the direction of Dr. Richard Youle of theNational Institute of Health has suggested that there would be anadvantage to modifying ranpirnase in a particular manner. Dr. Youle is apioneer in the field of “cysteinizing” therapeutically active RNases(specifically, human pancreatic RNase) with the object of increasingtheir effectiveness. Dr. Youle conceived the idea of re-engineering anRNase so it could be more easily attached to a targeting molecule,thereby making it possible for the RNase to be delivered to a particularcell receptor where it might be most effective. To achieve thisobjective, he utilized a property of the amino acid cysteine.

Cysteine has a single reactive sulfhydryl (“SH”) group. The availabilityof this group facilitates the chemical linking of a targeting moleculeto the cysteine residue. Dr. Youle realized that by conservativelysubstituting a cysteine residue at an appropriate location in an RNase,the RNase could easily be linked to a targeting moiety (such as amonoclonal antibody) that targets a predetermined cell receptor. Thiswould permit the RNase to be delivered to the precise location where itmight be most therapeutically effective.

Accordingly, it would be advantageous to produce a cysteinizedranpirnase, i.e. a modified ranpirnase in which an amino acid residue atan appropriate location was conservatively replaced by cysteine.

One object of the invention is to provide a nucleic acid that encodesranpirnase, and to provide a method of synthesizing that nucleic acid.

Another object of the invention is to provide a nucleic acid thatencodes cysteinized ranpirnase, and to provide a method of synthesizingthat nucleic acid.

In accordance with the invention, two nucleic acids are produced. Eachof these nucleic acids encodes a corresponding protein. One protein isconverted to ranpirnase by cleavage of an N-terminal methionine residueat position −1 and autocyclization of a glutamine residue at position 1.The other protein (after a like cleavage and autocyclization) isconverted to a cysteinized ranpirnase in which the methionine residue atposition 23 is replaced by a residue of leucine, and in which the serineresidue at position 72 is replaced by a residue of cysteine. (Thesubstitution of methionine at position 23 does not appear to adverselyaffect the bioactivity of the resulting RNase.) The cysteine residueprovides a location at which a targeting moiety (such as a monoclonalantibody) can be attached, to deliver the cysteinized ranpirnase to thatreceptor site where it can most efficiently be used.

In accordance with preferred embodiments of the invention, synthesis ofboth nucleic acids begins with a recombinant plasmid originallysynthesized in Dr. Youle's laboratory. This recombinant plasmid, namedpET11d-rOnc(Q1, M23L) and made up of a rOnc(Q1, M23L) gene cloned in apET-11d vector, encodes a protein. The protein is highly homologous toranpirnase, but a) has an N-terminal residue of methionine at position−1 followed by a residue of glutamine at position 1, and b) has aleucine residue at position 23 instead of a methionine residue (asranpirnase has). An overlapping PCR protocol is used to mutate therOnc(Q1, M23L) gene. In accordance with a first preferred embodiment ofthe invention, the rOnc(Q1, M23L) gene is modified to encode a proteinthat, after cleavage of its N-terminal methionine residue andautocyclization of an adjacent glutamine residue, is ranpirnase. Inaccordance with a second preferred embodiment of the invention, therOnc(Q1, M23L) gene is modified to encode a protein that, after a likecleavage and autocyclization, is a cysteinized ranpirnase wherein theleucine residue at position 23 is left in place and the serine residueat position 72 is changed to a residue of cysteine.

The expressed protein from each of these preferred embodiments has anN-terminal residue of methionine at position −1 followed by a residue ofglutamine at position 1. When in each instance the methionine residue iscut off (or “cleaved”), the glutamine autocyclizes to form pyroglutamicacid (which is also located at position 1 in ranpirnase).

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with the aid of the followingillustrative and non-limiting drawings, in which:

FIG. 1 is a flowchart showing a first preferred embodiment of theinvention; and

FIG. 2 is a flowchart showing a second preferred embodiment of theinvention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Ranpirnase, known by the trademark ONCONASE and presently undergoingclinical trials, has an amino acid sequence that is 104 residues longand that has pyroglutamic acid (<Glu) at its N-terminal. Thepyroglutamic acid is produced by autocyclization of glutamine (Gln)after the removal of a residue of methionine (Met) at position −1. Forthis reason, the amino acid sequence of ranpirnase is herein shown asSEQ ID NO:1.

A recombinant plasmid named pET11d-rOnc(Q1, M23L) and made up of therOnc(Q1,M23L) gene cloned in a pET-11d vector, encodes a protein thathas an N-terminal methionine residue at position −1, a residue ofglutamine at position 1, and that from position 2 on has the same aminoacid sequence as ranpirnase, except that in the encoded protein themethionine residue at position 23 is changed to a residue of leucine.(This recombinant plasmid was synthesized by YouNeng Wu in thelaboratory of Dr. Richard J. Youle, and is described in J. Mol. Biol.(1996) 257, 992-1007.) In accordance with a first preferred embodimentof the invention as is described immediately below, the pET11d-rOnc(Q1,M23L) gene is modified using an overlapping PCR protocol that changesthis position 23 leucine residue back to methionine (a “site-directedmutation”).

Reagents from Perkin Elmer (Branchburg N.J.), Stratagene (La JollaCalif.) and Novagen (Madison Wis.) were used for PCR and otherrecombinant DNA manipulations. The primers were designed to generateprotein fragments having an XbaI site at the 5′ end and a stop codonflanked by a BamHI site at the 3′ end. (The use of primers that generateXbaI and BamHI sites are not necessary and are not part of theinvention. These primers were chosen because the cloning vector isintended to be of pET-11d. If another vector were to be used, the XbaIand BamHI sites would be changed to the sites that are appropriate tothat other vector.)

In accordance with this first preferred embodiment, four primers areconstructed for use in an overlapping PCR protocol. These are a forwardPCR primer, a reverse PCR primer, a mutated forward PCR primer, and amutated reverse PCR primer, as follows: the forward PCR primer is SEQ IDNO:3, the reverse PCR primer is SEQ ID NO:4, the mutated forward PCRprimer is SEQ ID NO:5, and the mutated reverse PCR primer is SEQ IDNO:6. The forward PCR primer contains an XbaI restriction site, thereverse PCR primer contains a stop codon followed by a BamHI restrictionsite. The mutated forward and reverse PCR primers are chosen to carryout a particular site-directed mutation, namely to change the leucineresidue at position 23 to a residue of methionine.

In a first PCR reaction using Pfu DNA polymerase (FIG. 1, step 10), therecombinant plasmid pET11d-rOnc (Q1, M23L) DNA is used as a templatewith the forward PCR primer SEQ ID NO:3 and the mutated reverse PCRprimer SEQ ID NO:6. In a second PCR reaction using Pfu DNA polymerase(FIG. 1, step 20), the recombinant plasmid pET11d-rOnc(Q1, M23L) DNA isused as a template with the reverse PCR primer SEQ ID NO:4 and themutated forward PCR primer SEQ ID NO:5. These first and second PCRreactions produce overlapping DNA fragments that have the desiredmutation (leucine residue to methionine residue at location 23) in theirregions of overlap.

Then, in a third PCR reaction using Pfu DNA polymerase (FIG. 1, step30), these overlapping DNA fragments are mixed together with the forwardPCR primer SEQ ID NO:3 and the reverse PCR primer SEQ ID NO:4. Thisproduces a full-length gene having an XbaI restriction site at one endand a BamHI restriction site at the other. This new gene has been namedrOnc(Q1). The new rOnc(Q1) gene can then be cloned (FIG. 1, step 40)into a pET11d plasmid vector using the XbaI and BamHI restriction sites.The resulting pET11d-rOnc(Q1) recombinant plasmid is then used totransform an expression host cell (FIG. 1, step 50). An appropriate hostcell is E.coli BL21(DE3). A protein is expressed from the host cell(FIG. 1, step 60). The expressed protein has an N-terminal methionine(Met) residue at position −1 followed by a residue of glutamine (Gln) atposition 1. When the methionine residue is cleaved (FIG. 1, step 70) theglutamine autocyclizes to form pyroglutamic acid (<Glu), formingranpirnase (SEQ ID NO:1).

In a second preferred embodiment of the invention, the gene in thepET11d-rOnc(Q1, M23L) recombinant plasmid DNA is subjected to adifferent site-directed mutation. In this other site-directed mutation,the amino acid residue at position 72 in the encoded protein is changedfrom serine (Ser) residue to cysteine (Cys) using different overlappingPCR primers. In this different overlapping PCR protocol, the forward andreverse PCR primers (i.e. the primers that have the XbaI and BamHIrestriction sites) are the same as those used in the first preferredembodiment. However, the mutated forward and reverse PCR primers aredifferent, because they change the serine residue at position 72 in theencoded protein to cysteine.

Hence, in accordance with this second preferred embodiment, four primersare constructed for use in an overlapping PCR protocol. These are aforward PCR primer, a reverse PCR primer, a mutated forward PCR primer,and a mutated reverse PCR primer, as follows: the forward PCR primer isSEQ ID NO:3, the reverse PCR primer is SEQ ID NO:4, the mutated forwardPCR primer is SEQ ID NO:7, and the mutated reverse PCR primer is SEQ IDNO:8. As in the first preferred embodiment, the forward PCR primercontains an XbaI restriction site and the reverse PCR primer contains astop codon followed by a BamHI restriction site. However, in this secondpreferred embodiment, the mutated forward and reverse PCR primers arechosen to carry out a different site-directed mutation in the encodedprotein, namely a change of the residue at position 72 from serine tocysteine.

In a first PCR reaction using Pfu DNA polymerase (FIG. 2, step 80), therecombinant plasmid pET11d-rOnc(Q1, M23L) DNA is used as a template withthe forward PCR primer SEQ ID NO:3 and the mutated reverse PCR primerSEQ ID NO:8. In a second PCR reaction using Pfu DNA polymerase (FIG. 2,step 90), the recombinant plasmid pET11d-rOnc(Q1, M23L) DNA is used as atemplate with the reverse PCR primer SEQ ID NO:4 and the mutated forwardPCR primer SEQ ID NO:7. These first and second PCR reactions produceoverlapping DNA fragments that have the desired mutation (serine residueto cysteine residue at location 72) in their regions of overlap.

Then, in a third PCR reaction using Pfu DNA polymerase (FIG. 2, step100), these overlapping DNA fragments are mixed together with theforward PCR primer SEQ ID NO:3 and the reverse PCR primer SEQ ID NO:4.This produces a full-length gene having an XbaI restriction site at oneend and a BamHI restriction site at the other. This new gene has beennamed rOnc(Q1, M23L, S72C). The new rOnc (Q1, M23L, S72C) gene can thenbe cloned (FIG. 2, step 110) into a pET11d plasmid at the XbaI and BamHIrestriction sites to produce a pET11d-rOnc (Q1, M23L, S72C) recombinantplasmid. The resulting pET11d-rOnc (Q1, M23L, S72C) recombinant plasmidis then used to transform an E.coli BL21(DE3) host cell (FIG. 2, step120) to express the new rOnc (Q1, M23L, S72C) target gene (FIG. 2, step130). The expressed protein has an N-terminal methionine (Met) residueat position −1 followed by a residue of glutamine (Gln) at position 1.When the methionine residue is cleaved (FIG. 2, step 140) the glutamineautocyclizes to form pyroglutamic acid (<Glu), thereby formingcysteinized ranpirnase (SEQ ID NO:2).

Although at least one preferred embodiment of the invention has beendescribed above, this description is not limiting and is only exemplary.The scope of the invention is defined only by the following claims:

8 1 104 PRT Rana pipiens 1 Gln Asp Trp Leu Thr Phe Gln Lys Lys His IleThr Asn Thr Arg Asp 1 5 10 15 Val Asp Cys Asp Asn Ile Met Ser Thr AsnLeu Phe His Cys Lys Asp 20 25 30 Lys Asn Thr Phe Ile Tyr Ser Arg Pro GluPro Val Lys Ala Ile Cys 35 40 45 Lys Gly Ile Ile Ala Ser Lys Asn Val LeuThr Thr Ser Glu Phe Tyr 50 55 60 Leu Ser Asp Cys Asn Val Thr Ser Arg ProCys Lys Tyr Lys Leu Lys 65 70 75 80 Lys Ser Thr Asn Lys Phe Cys Val ThrCys Glu Asn Gln Ala Pro Val 85 90 95 His Phe Val Gly Val Gly Ser Cys 1002 104 PRT Artificial Sequence SEQ ID NO1 with Leu at position 23 and Cysat position 72 2 Gln Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn ThrArg Asp 1 5 10 15 Val Asp Cys Asp Asn Ile Leu Ser Thr Asn Leu Phe HisCys Lys Asp 20 25 30 Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val LysAla Ile Cys 35 40 45 Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr SerGlu Phe Tyr 50 55 60 Leu Ser Asp Cys Asn Val Thr Cys Arg Pro Cys Lys TyrLys Leu Lys 65 70 75 80 Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu AsnGln Ala Pro Val 85 90 95 His Phe Val Gly Val Gly Ser Cys 100 3 43 DNAArtificial Sequence Forward PCR Primer containing Xba1 site 3 caattcccctctagaaataa ttttgtttaa ctttaagaag gag 43 4 32 DNA Artificial SequenceReverse PCR Primer containing BamH1 site 4 cgcgcggatc cctactagcaagaaccaaca cc 32 5 39 DNA Artificial Sequence Mutated Forward PCR Primerto produce SEQ ID NO1 5 gactgcgaca acatcatgtc tactaacctg ttccattgc 39 639 DNA Artificial Sequence Mutated Reverse PCR Primer to produce SEQ IDNO1 6 gaacaggtta gtagacatga tgttgtcgca gtcaacgtc 39 7 39 DNA ArtificialSequence Mutated Forward PCR Primer to produce SEQ ID NO2 7 gactgcaacgttacttgccg tccgtgcaaa tacaaactg 39 8 39 DNA Artificial Sequence MutatedReverse PCR Primer to produce SEQ ID NO2 8 gtatttgcac ggacggcaagtaacgttgca gtcagacag 39

What is claimed is:
 1. An isolated nucleic acid encoding a proteinhaving an N-terminal methionine residue at position −1 and a glutamineresidue at position 1, said encoded protein, after its position −1methionine residue has been cleaved and its glutamine residue has beenautocyclized, being the Ribonuclease of SEQ ID NO:1.
 2. An isolatednucleic acid encoding a protein having an N-terminal methionine residueat position −1 and a glutamine residue at position 1, said encodedprotein, after its position −1 methionine residue has been cleaved andits glutamine residue has been autocyclized, being the Ribonuclease ofSEQ ID NO:2.
 3. A host cell expressing the nucleic acid of claim
 1. 4. Ahost cell expressing the nucleic acid of claim
 2. 5. A vector encodingthe Ribonuclease of SEQ ID NO:1.
 6. A vector encoding the Ribonucleaseof SEQ ID NO:2.
 7. The vector of claim 5, wherein the vector is aplasmid.
 8. The vector of claim 6, wherein the vector is a plasmid.